1. Field of the Invention
This invention relates to novel compositions comprising amino acid sequence variants of tissue factor protein. The tissue factor protein variants have a greater affinity for FVII/FVIIa than their mammalian tissue factor protein counterparts. The invention also relates to pharmaceutical compositions comprising the novel compositions as well as their use in diagnostic, therapeutic, and prophylactic methods.
2. Description of Related Disclosures
Tissue factor (TF) is the receptor for coagulation factor VIIa (FVIIa) and the zymogen precursor factor VII (FVII). TF is a 263 amino acid residue glycoprotein composed of a 219 residue extracellular domain, a single transmembrane domain, and a short cytoplasmic domain (Fisher et al., (1987) Thromb. Res. 48:89-99). The TF extracellular domain is composed of two immunoglobulin-like fibronectin type III domains of about 105 amino acids each. Each domain is formed by two anti-parallel xcex2-sheets with Ig superfamily type C2 homology. The protein interaction of FVIIa with TF is mediated entirely by the TF extracellular domain (Muller et al., (1994) Biochem. 33:10864-10870; Gibbs et al., (1994) Biochem. 33:14003-14010; Ruf et al., (1994) Biochem. 33:1565-1572) which has been expressed in E. coli, cultured Chinese Hamster Ovary (CHO) cells and Saccharomyces cerevisiae (Waxman et al., (1992) Biochemistry 31:3998-4003; Ruf et al., (1991) J. Bio. Chem. 266:2158-2166 and Shigamatsue et al., (1992) J. Biol. Chem. 267:21329-21337). The structures of the human TF (hTF) extracellular domain and its complex with active site inhibited FVIIa have recently been determined by x-ray crystallography (Harlos et al., (1994) Nature 370:662-666; Muller et al., (1994) Biochemistry 33:10864; Banner et al., (1996) Nature 380:41-46).
The hTF extracellular domain has also been extensively characterized by alanine scanning mutagenesis (Kelley et al., (1995) Biochemistry, 34:10383-10392; Gibbs et al., (1994) supra; Ruf et al., (1994) supra). Residues in the area of amino acids 16-26 and 129-147 contribute to the binding of FVIIa as well as the coagulant function of the molecule. Residues Lys20, Trp45, Asp58, Tyr94, and Phe140 make a large contribution (1 kcal/mol) to the free energy (xcex94G) of binding to FVIIa (Kelley et al., (1995) supra). Substitution of Lys20 and Asp58 with alanine residues leads to 78- and 30-fold reductions in FVIIa affinity respectively (Kelley et al., (1995) supra). A set of 17 single-site mutants at other nearby sites that are in contact with FVIIa result in modest decreases in affinity (xcex94xcex94G=0.3-1.0 kcal molxe2x88x921). Mutations of TF residues Thr17, Arg131, Leu133 and Val207, each of which contact FVIIa in the crystal structure, have no effect on affinity for FVIIa. Lys15Ala and Tyr185Ala mutations result in small increases in affinity (xcex94xcex94G=xe2x88x920.4 kcal molxe2x88x921) (Kelley et al., (1995) supra). The 78-fold decrease in affinity imposed by the alanine substitution of Lys20 in hTF can be reversed by substituting a tryptophan for Asp58 (Lee and Kelley, (1998) J. Biol. Chem. 273:4149-4154).
Residues in the area of amino acids 157-168 contribute to the procoagulant function of TF-FVIIa (Kelley et al., (1995) supra; Ruf et al., (1992) J. Biol. Chem. 267:22206-22210) but are not important for FVII/FVIIa binding. It has been shown that lysine residues 165 and 166 are important to TF cofactor function but do not participate in FVIIa complex formation (Roy et al., (1991) J. Biol. Chem. 266:22063; Ruf et al., (1992) J. Biol. Chem. 267:6375). Lysine residues 165 and 166 are located on the C-terminal fibronectin type III domain of TF on the opposite surface of the molecule from residues found to be important for FVIIa binding on the basis of mutagenesis results (Kelley et al., (1995) supra). Alanine substitution of these lysine residues results in a decreased rate of FX activation catalyzed by the TF-FVIIa complex (Ruf et al., (1992) supra). The Lys165Ala-Lys166Ala variant (hTFAA) comprising residues 1-219 of hTF (sTF) inhibits the extrinsic pathway of blood coagulation in vitro through competition with membrane TF for binding to FVIIa. In a rabbit model of arterial thrombosis the variant partially blocks thrombus formation without increasing bleeding tendency (Blood 89, 3219-3227). However, high doses of the variant are required for the antithrombotic effect, in part because FVIIa binds to cell surface TF approximately 1000-fold more tightly than to sTF (Kelley et al. (1997) supra). The greater apparent affinity is due to interaction of the FVIIa xcex3-carboxyglutamic acid-containing (Gla) domain with phospholipid.
TF is expressed constitutively on cells separated from plasma by the vascular endothelium (Carson, S. D. and J. P. Brozna, (1993) Blood Coag. Fibrinol. 4:281-292). Its expression on endothelial cells and monocytes is induced by exposure to inflammatory cytokines or bacterial lipopolysaccharide (Drake et al., (1989) J. Cell Biol. 109:389). Upon tissue injury, the exposed extracellular domain of TF forms a high affinity, calcium dependent complex with FVII. Once bound to TF, FVII can be activated by peptide bond cleavage to yield serine protease FVIIa. The enzyme that catalyzes this step in vivo has not been elucidated, but in vitro FXa, thrombin, TF-FVIIa and FIXa can catalyze this cleavage (Davie, et al., (1991) Biochem. 30:10363-10370). FVIIa has only weak activity upon its physiological substrates FX and FIX whereas the TF-FVIIa complex rapidly activates FX and FIX.
The TF-FVIIa complex constitutes the primary initiator of the extrinsic pathway of blood coagulation (Carson, S. D. and Brozna, J. P., (1993) Blood Coag. Fibrinol. 4:281-292; Davie, E. W. et al., (1991) Biochemistry 30:10363-10370; Rapaport, S. I. and L. V. M. Rao, (1992) Arterioscler. Thromb. 12:1111-1121). The complex initiates the extrinsic pathway by activation of FX to Factor Xa (FXa), FIX to Factor IXa (FIXa), and additional FVII to FVIIa. The action of TF-FVIIa leads ultimately to the conversion of prothrombin to thrombin, which carries out many biological functions (Badimon, L. et al., (1991) Trends Cardiovasc. Med. 1:261-267). Among the most important functions of thrombin is the conversion of fibrinogen to fibrin, which polymerizes to form a clot. The TF-FVIIa complex also participates as a secondary factor in extending the physiological effects of the contact activation system.
The involvement of this plasma protease system has been suggested to play a significant role in a variety of clinical manifestations including arterial and venous thrombosis, septic shock, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC) and various other disease states (Haskel, E. J. et al., (1991) Circulation 84:821-827); Holst, J. et al., (1993) Haemostasis 23 (suppl. 1):112-117; Creasey, A. A. et al., (1993) J. Clin. Invest. 91:2850-2860; see also, Colman R. W. (1989) N. Engl. J. Med 320:1207-1209; Bone, R. C. (1992) Arch. Intern. Med. 152:1381-1389). Overexpression and/or aberrant utilization of TF has been linked to the pathophysiology of both thrombosis and sepsis (Taylor et al., (1991) Circ. Shock 33:127; Warr et al., (1990), Blood 75:1481; Pawashe et al., (1994) Circ. Res. 74:56). TF is expressed on cells found in the atherosclerotic plaque (Wilcox et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:2839). Additionally, TF has been implicated in tumor metastasis (Bromberg et al., (1995) Proc. Natl. Acad. Sci., USA, 92:8205). Neutralizing anti-TF monoclonal antibodies have been shown to prevent death in a baboon model of sepsis (Taylor et al., (1991) Circ. Shock 33:127), attenuate endotoxin-induced DIC in rabbits (Warr et al., (1990), Blood 75:1481), and to prevent thrombus reformation in a rabbit model of arterial thrombosis (Pawashe et al., (1994) Circ. Res. 74:56).
The present invention provides compositions comprising amino acid sequence variants of tissue factor protein. The tissue factor protein variants have a greater affinity for FVII/FVIIa than mammalian tissue factor protein counterparts from which they are derived. In preferred embodiments, the present invention provides compositions which inhibit a TF-FVIIa mediated or associated process such as the catalytic conversion of FVII to FVIIa, FIX to FIXa, or FX to FXa and thereby block initial events of the extrinsic pathway of blood coagulation. Accordingly, the present invention provides tissue factor protein variants that are optionally defective as cofactors for coagulation factor X activation. Therefore, the compositions of the present invention are capable of competing with endogenous tissue factor for binding to FVII or FVIIa and, according to certain aspects, capable of neutralizing the thrombotic effects of endogenous tissue factor.
The compositions of the present invention are useful in therapeutic and prophylactic methods for treating bleeding disorders. For example, according one aspect of the invention the tissue factor protein variant is formulated as a coagulation-inducing therapeutic composition for various chronic and acute bleeding disorders including deficiencies of coagulation factors VIII, IX or XI. According to a further aspect, the invention provides therapeutic and prophylactic methods as well as compositions for inhibiting TF-FVIIa mediated or associated processes. Advantageously, the compositions provide for low dose pharmaceutical formulations.
According to particular aspects of the present invention, a tissue factor protein variant is provided having an amino acid sequence derived from a mammalian tissue factor protein wherein at least one amino acid residue corresponding to a human amino acid residue selected from the group consisting of Asp54, Glu56, Glu130, Arg131, Leu133, Arg135 and Phe140 is substituted with another amino acid, the tissue factor protein variant having a greater affinity for FVII/FVIIa than the mammalian tissue factor protein from which it is derived. Preferably, the tissue factor protein variant is a soluble tissue factor protein variant having at least one amino acid residue selected from the group consisting of Asp54 and Glu56, and at least one amino acid residue selected from the group consisting of Glu130, Arg131, Leu133, Arg135 and Phe140 substituted with another amino acid. According to particular aspects of the invention, the other amino acid residue for Asp54 is preferably selected from the group consisting of Lys, Asn, Glu, Ala and Ser; the other amino acid residue for Glu56 is preferably selected from the group consisting of Asp, His, Gln and Trp; the other amino acid residue for Glu130 is preferably selected from the group consisting of Asp, Ala, Ser and Gly, the other amino acid residue for Arg131is preferably selected from the group consisting of Gln, Ile, Pro, Ser, Leu, Lys, Thr and Met, the other amino acid residue for Leu133 is preferably Ala, the other amino acid residue for Arg135 is preferably selected from the group consisting of Trp, Gln, Leu, Tyr, Thr, and Ala and the other amino acid residue for Phe140 is preferably selected from the group consisting of Asn, His, Val, Ala, Arg and Gly.
The present invention additionally provides for tissue factor protein variants having further amino acid substitutions at amino acid residues which contribute energetically to Factor VII/VIIa binding or which contribute to FVII/FVIIa cofactor activity. Accordingly, the invention provides amino acid sequence variants of tissue factor protein which are defective in FVIIa cofactor function and which have an increased affinity for FVII/FVIIa compared with counterpart tissue factor proteins. According to a particular aspect of the invention at least one additional amino acid residue, preferably selected from the group consisting of Lys15, Asp44, Trp158, Ser163, Gly164, Lys165, Lys166 and Tyr185 is substituted with another amino acid residue such as alanine.
In one embodiment, the composition of the present invention is a polypeptide and the invention encompasses a composition of matter comprising an isolated nucleic acid, preferably DNA, encoding the polypeptide of the invention. According to this aspect, the invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA molecule, where the control sequence is recognized by a host cell transformed with the vector, and a host cell transformed with the vector.
The present invention further extends to therapeutic applications for the compositions described herein. Thus the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the composition of the invention. Pharmaceutical compositions comprising these molecules can be used in the treatment or prophylaxis of thrombotic or coagulopathic related diseases or disorders including hereditary deficiencies in coagulation factors, vascular diseases and inflammatory responses. These applications include, for example, a method of treating a mammal for which inhibiting TF-FVIIa is indicated comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the mammal. Such indications include; deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angioplasty (PTCA), stroke, tumor metastasis, inflammation, septic shock, hypotension, ARDS, and DIC. The compositions of the present invention may also be used as an adjunct in thrombolytic therapy.